Deep profiling of potential substrate atlas of porcine epidemic diarrhea virus 3C-like protease.

Coronavirus (CoV) 3C-like protease (3CLpro) is essential for viral replication and is involved in immune escape by proteolyzing host proteins. Deep profiling the 3CLpro substrates in the host proteome extends our understanding of viral pathogenesis and facilitates antiviral drug discovery. Here, 3CLpro from porcine epidemic diarrhea virus (PEDV), an enteropathogenic CoV, was used as a model which to identify the potential 3CLpro cleavage motifs in all porcine proteins. We characterized the selectivity of PEDV 3CLpro at sites P5-P4'. We then compiled the 3CLpro substrate preferences into a position-specific scoring matrix and developed a 3CLpro profiling strategy to delineate the protein substrate landscape of CoV 3CLpro. We identified 1,398 potential targets in the porcine proteome containing at least one putative cleavage site and experimentally validated the reliability of the substrate degradome. The PEDV 3CLpro-targeted pathways are involved in mRNA processing, translation, and key effectors of autophagy and the immune system. We also demonstrated that PEDV 3CLpro suppresses the type 1 interferon (IFN-I) cascade via the proteolysis of multiple signaling adaptors in the retinoic acid-inducible gene I (RIG-I) signaling pathway. Our composite method is reproducible and accurate, with an unprecedented depth of coverage for substrate motifs. The 3CLpro substrate degradome establishes a comprehensive substrate atlas that will accelerate the investigation of CoV pathogenicity and the development of anti-CoV drugs.IMPORTANCECoronaviruses (CoVs) are major pathogens that infect humans and animals. The 3C-like protease (3CLpro) encoded by CoV not only cleaves the CoV polyproteins but also degrades host proteins and is considered an attractive target for the development of anti-CoV drugs. However, the comprehensive characterization of an atlas of CoV 3CLpro substrates is a long-standing challenge. Using porcine epidemic diarrhea virus (PEDV) 3CLpro as a model, we developed a method that accurately predicts the substrates of 3CLpro and comprehensively maps the substrate degradome of PEDV 3CLpro. Interestingly, we found that 3CLpro may simultaneously degrade multiple molecules responsible for a specific function. For instance, it cleaves at least four adaptors in the RIG-I signaling pathway to suppress type 1 interferon production. These findings highlight the complexity of the 3CLpro substrate degradome and provide new insights to facilitate the development of anti-CoV drugs.